The purpose of this study was to determine the effect of the oral administration of pyridostigmine bromide on indices of heart rate variability (HRV) in healthy young volunteers. Seventeen healthy participants (11 men, 6 women; aged 27±8 y) submitted to a randomized, crossover, double-blind protocol, in which they received 30 mg pyridostigmine bromide (PYR) or placebo orally at 8-hour intervals for 24 hours, on two separate days. Venous blood samples were collected 2 and 24 hours after the first dose for determination of serum cholinesterase activity. Holter tapes were recorded during the 24-hour period and analyzed using a semiautomatic technique to evaluate time- and frequency-domain indices of HRV and to build three-dimensional return maps for later quantification. Symptoms were mild and occurred similarly during administration of PYR and placebo (p=0.140). Serum cholinesterase activity was reduced by 15% at 2 hours (p=0.013) and by 14% at 24 hours (p=0.010) after the first dose of PYR, but not after administration of placebo. Pyridostigmine administration caused a significant increase in the mean 24-hour R-R interval (placebo: 814±20 msec; PYR: 844±18 msec; p=0.003) and in time-domain indices of HRV, such as the standard deviation of all R-R intervals (SDNN; placebo: 151±9 msec; PYR: 164 ±9 msec; p=0.017), and the percentage of pairs of adjacent R-R intervals differing by more than 50 msec (pNN50; placebo: 12.8±1.8%; PYR: 13.9±1.5%; p=0.029). Pyridostigmine had no significant effect on frequency-domain indices of HRV, but resulted in significant increase in P₂, a parasympathetic index derived from the three-dimensional return map (placebo: 93±13 msec; PYR: 98±13 ms; p=0.029). In conclusion, low-dose pyridostigmine reduced mean heart rate and increased HRV during a 24-hour period in healthy young subjects.